This invention relates to novel vaccine compositions, processes for the preparation thereof and methods for the use thereof for the mucosal (nonparenteral) immunization of warm blooded animals.
There are two main types of immunity which may provide a host organism protection against disease and/or infection: systemic (or general) immunity which is provided through parenteral vaccination; and mucosal (or local) immunity which is provided through nonparenteral vaccination.
Traditionally, vaccine development has focused on the induction of systemic immunity, including humoral (specific antibodies of IgM or IgG class) and cellular immune responses (activated T lymphocytes, activated macrophages, or others) through the use of parenteral vaccines. Such parenteral vaccines are administered through, inter alia, intramuscular or subcutaneous routes.
While providing systemic immunity through the use of parenteral vaccines and parenteral vaccination has been proven to be effective in establishing protective immunity against many different pathogens, this is not always the case. Numerous examples exist where such immunity has been proven to be wholly or partially ineffective. Furthermore, parenteral vaccines and parenteral vaccination to provide systemic immunity has other disadvantages, such as the need to breach the integrity of the skin of the organism being immunized therewith, difficulty of administration (needing, for example, trained personnel to administer such vaccines), the high grade of purity needed for such vaccines, a lack of establishment of immunity at the site of natural infection, nonprevention of infection and less than complete protection of the organism against both clinical and nonclinical symptoms of the infection as well as against the infection itself. Further, parenteral vaccines can present problems where the effective immunization of immnunocompromised hosts (i.e., young animals with maternal antibodies) is desired.
A multitude of pathogens naturally infect their hosts via the mucosal (e.g. the respiratory, gastrointestinal or genital) tissues. Mucosal (or local) immunity results from the local formation and secretion of antibodies of the IgA class. These antibodies form dimers which can be secreted into the lumen of respiratory, gastro-intestinal or genital organ. Specific IgA antibodies in the lumen are capable of reducing infection by impairing or blocking penetration of the host tissue by the pathogen. Mechanisms known to underly the inhibition of host tissue penetration include: the neutralization of viruses; the complexation with enzymes, toxins or other components produced by the pathogens (resulting in either neutralization of the activity of these components and/or blocking the adsorption of these components); the inhibition of adherence of the pathogens to mucosal surfaces; the suppression of antibody mediated inflammnatory reactions at the mucosal surfaces; and synergism with innate antibacterial factors at the musocal surface.
Mucosal (nonparenteral) vaccines and mucosal vaccination have other additional advantages over parenteral vaccines and parenteral vaccination. These advantages include the elimination of the need to breach the integrity of the skin, tissues or organs of the host, ease of administration, the possibility of employing a low grade of purity, establishment of immunity at the site of natural infection, prevention of penetration of the host tissue by the pathogen, more complete protection of the organism against both clinical and nonclinical symptoms of the infection as well as the infection itself, protection against latency and concomitant induction of mucosal and systemic immunity. Furthermore, mucosal vaccines and mucosal vaccination permit the effective immunization of immunocompromised hosts (i.e., young animals with maternal antibodies).
Thus, it can be seen that, in many cases the use of mucosal (nonparenteral) vaccines, mucosal vaccination and the immunity provided thereby is preferable over the use of parenteral vaccines, parenteral vaccination and the immunity provided thereby.
Depending on various factors, natural or artificial infection with live microorganisms can induce considerable levels of mucosal immunity. These factors include: the route of infection, the nature of the microorganism, the infectious dose involved and the immune status of the host.
However, the administration of killed (non-replicating) antigens gives little or no mucosal immunity. To alleviate this problem, adjuvants are used to increase the immune responses to killed antigens.
The adequate induction of mucosal immunity with killed (non-replicating) antigens requires both the administration of antigen to the mucosae and the use of appropriate adjuvants or antigen presentation systems.
While numerous adjuvants for parenteral vaccines are known, only a few have been shown to be useful in enhancing mucosal immunity. Such adjuvants include the toxin of Vibrio cholera (Cholera toxin) or products thereof (Cholera toxin subunit Bxe2x80x94CTB), the heat-labile toxin of E. coli or products thereof, bacterial toxins or products thereof which are conjugated to antigens, microparticles or microcapsules of different natural or synthetic polymers having antigens incorporated therein, liposomes antigen incorporated therein or liposomes mixed with antigen, lectins, immunostimnulating complexes, muramyldipeptide and derivatives thereof, and cationic polymers (see, xe2x80x9cNovel Delivery Systems for Oral Vaccinesxe2x80x9d, CRC Press, London, 1994).
Although well-known, the use of known mucosal adjuvants has been limited. This has been due to several factors, including: the unacceptable risks associated with the detrimental side effects of such adjuvants; problems of insufficient efficacy; the (partial) denaturation of antigens resulting from mechanical and/or chemical treatments which are involved in their preparation procedure; complicated production procedures which are associated therewith; the inconsistency of the production thereof, the high costs of their production; specific immune responses elicited to the adjuvant component; the instability of the adjuvant or the vaccine containing the adjuvant; and the enhancement or activation of nonspecific immune reactions which results from the use thereof.
Thus it can be seen that there remains an urgent need for adjuvants for mucosal vaccines (and, in particular for mucosal vaccines against respiratory diseases, gastro-intestinal diseases and sexually-transmissible diseases) which are safe, inexpensive, easy to produce and to incorporate into mucosal vaccines.
It is a primary object of the present invention to provide mucosal adjuvants capable of inducing or enhancing immune responses to antigens.
It is a further primary object of the present invention to provide mucosal adjuvants for mucosal vaccines (and, in particular, for mucosal vaccines against respiratory diseases, gastro-intestinal diseases and sexually-transmissible diseases) which are safe, inexpensive, easy to produce and easy to incorporate in to such mucosal vaccines in which they are to be employed.
It is a still further primary object of the present invention to provide mucosal vaccines which incorporate such mucosal adjuvants therein for inducing or enhancing immune responses to antigens.
It is a yet further primary object of the present invention to provide methods for inducing or enhancing immune responses to antigens and to provide methods for providing mucosal adjuvants and mucosal vaccines comprised of mucosal adjuvants which are capable of such inducement or enhancement.
The present invention relates to mucosal adjuvants for incorporation into mucosal vaccines and to mucosal vaccines incorporating such adjuvants therein useful for the induction or enhancement of mucosal and/or systemic immune responses to antigens.
Thus, in accordance with the teachings of the present invention, there is provided a mucosal adjuvant for vaccines comprising a water-soluble polyanionic polymer having anionic constitutional repeating units.
The mucosal adjuvants of the present invention are water-soluble polyanionic polymers having anionic constitutional repeating units which may be the same or different repeating units, or polyanionic polymers having anionic constitutional repeating units (same or different) and hydrophobic constitutional repeating units. The polyanionic polymers may be linear (polymers having chemical units which are connected covalently to one or two other constitutional units), or branched (polymers having chemical units which are connected covalently to one or two other constitutional units and occasionally to three or more constitutional units) or reticular (polymers having chemical units which are connected covalently to one or two or three or more other constitutional units) in structure.
As used herein, the following terms have the following meanings:
The term xe2x80x9cwater solublexe2x80x9d, when referring to the polyanionic polymers of the present invention refers to polymers which are soluble in an aqueous phase at a concentration of at least 0.01 gram per liter.
The term xe2x80x9cpolymer refers to compounds having at least three identical chemical constitutional repeating units, which said units are covalently connected with one another.
The term xe2x80x9cconstitutional repeating unit refers to the minimal structural unit of a polymer.
The term xe2x80x9chomopolymerxe2x80x9d refers to polymers consisting of one type of constitutional repeating unit.
The term xe2x80x9cheteropolymerxe2x80x9d refers to polymers having two or more different constitutional repeating units.
The term xe2x80x9cpolyanionic polymerxe2x80x9d refers to polymers which, when dissolved in an aqueous medium, are negatively charged due to the presence of anionic constitutional repeating units (e.g., units containing sulphate, sulphonate, carboxylate, phosphate and borate groups).
The term xe2x80x9canionic constitutional repeating unitxe2x80x9d refers to constitutional repeating units of polymers which are negatively-charged in aqueous medium at physiological conditions.
The term xe2x80x9chydrophobic constitutional repeating unitxe2x80x9d refers to constitutional repeating units of polymers which are characterised in that the corresponding monomer is less soluble in an aqueous phase than in an organic solvent [that is to say, the quantity, in weight (in grams), of the monomer that can be dissolved in a fixed volume, in ml, of an aqueous phase is less than the quantity, in weight (in grams), of the monomer that can be dissolved in the same fixed volume, in ml, of the organic solvent].
In the mucosal adjuvants of the present invention the anionic constitutional repeating units of the polyanionic polymer are preferably obtained from acrylic acid, methacrylic acid, maleic acid, fumaric acid, ethylsulphonic acid, vinylsulphuric acid, vinylsulphonic acid, styrenesulphonic acid (vinylbenzenesulphonic acid), vinylphenylsulphuric acid, 2-methacryloyloxyethane sulphonic acid, 3-methacryloyloxy-2-hydroxypropanesulphonic acid, 3-methacryl amido-3-methylbutanoic acid, acrylamidomethylpropanesulfonic acid, vinylphosphoric acid, 4-vinylbenzoic acid, 3-vinyl oxypropane-1-sulphonic acid, N-vinylsuccinimidic acid, and salts of the foregoing.
More preferably, in the mucosal adjuvants of the present invention, the anionic constitutional repeating units of the polyanionic polymer are obtained from acrylic acid, methacrylic acid, maleic acid, fumaric acid, ethylsulphonic acid, vinylsulphuric acid, vinylsulphonic acid, styrenesulphonic acid, and acrylamidomethylpropanesulfonic acid, and salts of the foregoing.
Most preferred in the mucosal adjuvants of the present invention, the anionic constitutional repeating units of the polyanionic polymer are obtained from acrylic acid, methacrylic acid, maleic acid and fumaric acid, vinylsulphonic acid, styrenesulphonic acid, and acrylamidomethylpropanesulfonic acid, and salts of the foregoing.
The mucosal adjuvants of the present invention include polyanionic homopolymers which are preferably obtained from polyacrylic acid, polymethacrylic acid, polymaleic acid, polyfumaric acid, polyethylsulphonic acid, polyvinylsulphuric acid, polyvinylsulphonic acid, polystyrenesulphonic acid (polyvinylbenzenesulphonic acid), polyvinylphenylsulphuric acid, poly 2-methacryloyloxyethanesulphonic acid, poly 3-methacryloyloxy-2-hydroxypropanesulphonic acid, poly 3-methacryl amido-3-methylbutanoic acid, polyacrylamidomethylpropanesulfonic acid, polyvinylphosphoric acid, poly 4-vinylbenzoic acid, poly 3-vinyl oxypropane-1-sulphonic acid, poly N-vinylsuccinimidic acid, and salts of the foregoing.
More preferably, the polyanionic homopolymers are obtained from polyacrylic acid, polymethacrylic acid, polymaleic acid, polyfumaric acid, and salts of any of the fore going.
The mucosal adjuvants of the present invention further include polyanionic heteropolymers having two different (distinct) anionic groups, such as, but not limited to, a carboxylic group and a sulfate or sulfonic group, for example, acrylic acid and any of vinylsulphonic acid, styrenesulphonic acid and acrylamidomethylpropanesulfonic acid.
In a preferred embodiment, the polyanionic polymer of the mucosal adjuvants of the present invention further has hydrophobic constitutional repeating units.
The hydrophobic constitutional repeating units of the polyanionic polymer of the mucosal adjuvants of the present invention are obtained from alkylesters, cycloalkylesters, hydroxyalkylesters, ethers, glycols and aromatic groups and salts of the foregoing.
Preferably, in the mucosal adjuvants of the present invention, the alkylesters are selected from the group consisting of methyl-, ethyl-, propyl-, isopropyl, n-butyl-, isobutyl, sec.butyl-, t-butyl, n-hexyl-, n-octyl-, isooctyl-, 2-ethylhexyl-, n-decyl-, tetradecyl-, vinyl-, allyl- and oleylester.
Preferably, in the mucosal adjuvants of the present invention, the cycloalkylesters are selected from the group consisting of cyclohexyl-, 1-methylcyclohexyl-, 3-vinylcyclohexyl- and 3,3,5-trimethylcyclohexylester.
Preferably, in the mucosal adjuvants of the present invention, the hydroxyalkylesters are selected from the group consisting of 2-hydroxyethyl-, 2-hydroxypropyl-, 3-hydroxypropyl-, 3,4-dihydroxybutyl-, 2-hydroxypenyl- and 2-hydroxyhexylester.
Preferably, in the mucosal adjuvants of the present invention the ethers are selected from the from the group consisting of methoxymethyl, ethoxyethyl, allyloxymethyl, 2-ethoxyethoxymethyl, benzyloxymethyl, cyclohexyloxymethyl, 1-ethoxyethyl, 2-ethoxyethyl, 2-butoxyethyl, methoxymethoxyethyl, methoxyethoxyethyl, 1-butoxypropyl, 1-ethoxybutyl, tetrahydrofurfuryl, furfuryl.
Preferably, in the mucosal adjuvants of the present invention, the glycols are selected from the group consisting of ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,3-butanediol, 1,4-butanediol, 2,5-dimethyl- 1,6-hexanediol, 1,10-decanediol, diethyleneglycol and triethyleneglycol.
Preferably, in the mucosal adjuvants of the present invention, the aromatic groups are selected from the group consisting of benzyl, phenyl and nonylphenyl.
More preferably, in the mucosal adjuvants of the present invention the hydrophobic constitutional repeating units of the polyanionic polymer are obtained from the group consisting of methyl-, ethyl, propyl, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, and decyl- esters of acrylic acid, methacrylic acid, maleic acid, fumaric acid, ethylsulphonic acid, vinylsulphuric acid and styrenesulphonic acid and salts of the foregoing.
Most preferably, in the mucosal adjuvants of the present invention the hydrophobic constitutional repeating units of the polyanionic polymer are obtained from the group consisting of butyl-, pentyl-, hexyl-, heptyl- and octyl- esters of acrylic acid, methacrylic acid, maleic acid and fumaric acid and salts of the foregoing.
Particulaly preferred polyanionic polymers according to the present invention are polyacrylic acid, butyl-polyacrylic acid, poly(acrylate-co-acrylamidomethylpropane sulfonic acid) copolymer (p(A-c-AMPS)), poly(acrylate-co-vinylsulfonate) copolymer (p(A-c-VS)), poly(acrylate-co-vinylbenzenesulfonate) copolymer (p(A-c-VBS)),
It is further preferred that in the mucosal adjuvants of the present invention, the molar ratio of hydrophobic constitutional repeating units and anionic constitutional repeating units of the polyanionic polymers of the present invention is between 0 hydrophobic constitutional repeating units per 1 anionic constitutional repeating unit and 0.6 hydrophobic constitutional repeating units per 1 anionic constitutional repeating unit.
More preferably, in the mucosal adjuvants of the present invention, the molar ratio of hydrophobic constitutional repeating units and anionic constitutional repeating units is between 0.02 and 0.60 hydrophobic constitutional repeating unit per 1 anionic constitutional repeating unit (which is from 2 to 60 hydrophobic constitutional repeating units per every 100 anionic constitutional repeating units).
Most preferably, in the mucosal adjuvants of the present invention, the molar ratio of hydrophobic constitutional repeating units and anionic constitutional repeating units is between 0.05 and 0.30 hydrophobic constitutional repeating units per 1 anionic constitutional repeating unit (which is from 5 to 30 hydrophobic constitutional repeating units per every 100 anionic constitutional repeating units).
In another aspect of the present invention, disclosed herein is a mucosal (nonparenteral) vaccine having the mucosal adjuvant (including the polyanionic polymer thereof) of the present invention, wherein the vaccine is administered nonparenterally for the induction of either systemic or mucosal immunity against antigens.
In this aspect, the mucosal vaccine may further be comprised of an antigen or a drug molecule and/or a pharmaceutically-acceptable medium (carrier).
In yet another aspect of (lie present invention, disclosed herein is the use of the water-soluble polyanionic polymers of the present invention for the manufacture or production of mucosal adjuvants for the inducement or enhancement of mucosal or systemic immune responses.
In still another aspect of the present invention, the nonparenteral adjuvant comprised of a polyanionic polymer disclosed herein is administered nonparenterally for the enhancement of either systemic or mucosal immunity against antigens.
The antigen includes live or inactivated viruses, bacteria, fungi, parasites and other microorganisms as well as components or products derived from these microorganisms, products obtained by chemical synthesis capable of eliciting protective immunity, and products obtained by any other means capable of eliciting protective immunity.
Preferred antigens are those capable of eliciting protective immunity to diseases which are infections of the respiratory tract. Examples of such diseases are Newcastle disease virus, infectious bronchitis virus, influenza virus, rhinovirus, parainfluenza virus, adenovirus, Actinobaccilus pleuropneumoniae, Pasteurella multocida, Streptococcus pneumonia, Streptococcus pyogenes, and infections of the gastro-intestinal tract with for example, rotavirus, parvovirus, caronavirus, E. coli, Salmonella, Shigella, Yersinia, Campylobactor, Clostridium, Vibrio and Giardia, Entamoeba, and Cryptosporidium.
The polyanionic polymers which may be utilized as the adjuvants of the present invention may be obtained either by the isolation and purification of natural forms thereof or by the synthesis thereof.
Methods to synthesize polyanionic polymers having anionic constitutional repeating units and/or both anionic constitutional repeating units and hydrophobic constitutional repeating units are well known in the state of the art. Such methods include: copolymerisation of anionic and hydrophobic monomers; direct (partial) grafting of appropriate polymers; indirect (partial) grafing of appropriate polymers; and partial hydrolysis of appropriate polymers.
The use of the adjuvants of the present invention in vaccines for mucosal vaccination (immunization) offers various important advantages over those known mucosal adjuvants (and over parenteral vaccines) in that the adjuvants of the present invention are inexpensive, nonimmunogenic, water-soluble, chemically stable, easy to produce and easy to incorporate in the vaccines in which they are intended to be used. Furthermore, these adjuvants are more effective in inducing the desired mucosal immune responses than other adjuvants of which we are aware. Finally, various of the adjuvants described herein are already widely applied in food and pharmaceutical preparations, thereby increasing their acceptability.
The observations relative to the mucosal adjuvants of the present invention, and the mucosal vaccines which incorporate such mucosal adjuvants therein which are illustrated in the examples set forth below are considered unexpected in that: most well-known adjuvants for systemic immunity are not effective in enhancing mucosal immunity; those mucosal adjuvants which are presently employed are moderate or poor adjuvants for systemic immunity; there are crucial differences which exist in the mechanisms that are involved in the induction and development of systemic and mucosal immunity; and the polyanionic polymers disclosed herein are more effective than several of the most-promising mucosal adjuvants presently known.
It is further noted that the polyanionic polymers of the present invention are useful for the induction and/or enhancement of mucosal immune responses to antigens when they are administered either in conjunction with the antigen or separately from the antigen via nonparenteral routes.
The mucosal vaccines having the adjuvants of the present invention are effective for the induction of mucosal immunity, and include both an antigen and an adjuvant, wherein the adjuvant is a water-soluble polyanionic polymer.
The adjuvants of the present invention are solids (for example, are in the form of a powder). If desired, they may be used as such, being applied directly to the surface where an immune response is desired. In such case, being water-soluble, they are solubilized by the mucosal surfaces natural liquids.
Alternatively, the mucosal adjuvants of the present invention may be incorporated into an aqueous solution by being dissolved or incorportade into a liquid medium.
In this regard, the mucosal adjuvants of the present invention may further be incorporated into a vaccine having a liquid medium (such as a pharamaceutically-acceptable carrier). This may be achieved by, for example, being solubilized (as, for example, a powder) in a solution (such as an aqueous solution) containing, for example, an antigen (and/or a drug molecule). Another alternative method of achieving this may be by first dissolving the solid adjuvant in an aqueous phase which may then be either mixed with an aqueous solution of the antigen (and/or drug molecule) or which may then have a lyophilized antigen (and/or drug molecule) solubilized in the solution containing the adjuvant.
Preferably, the vaccines of the present invention are formulated so as to have between 0.01 and 40 mg of the polyanionic polymer (mucosal adjuvant) per ml vaccine.
More preferred, the vaccines of the present invention are formulated so as to have between 0.02 and 20 mg of the polyanionic polymer (mucosal adjuvant) per ml vaccine.
Most preferably the vaccines of the present invention are formulated so as to have between 0.25 and 5 mg of the polyanionic polymer (mucosal adjuvant) per ml vaccine.
The vaccines of the present invention may be applied to mucosal surfaces of animals or humans by nonparenteral routes such as intranasal, oral, oro-nasal, intratracheal and intracloacal. Such application may be made by, for example, the use of liquid aerosols, drinking-water, food, etc.
As used herein, the following terms shall have the meanings which are given therefor:
The term xe2x80x9cparenteral immunizationxe2x80x9d means the administration of a vaccine via the skin by use of a needle or another device using, inter alia one of the following routes: intracutaneous, subcutaneous, intraperitoneal, intramuscular and/or intradermal.
The terms xe2x80x9cnonparenteral immunizationxe2x80x9d and xe2x80x9cmucosal immunizationxe2x80x9d refer to the administration of a vaccine to a mucosal surface by, inter alia one of the following routes: intranasal, oro-nasal, intratracheal, intragastrie, intra-testinal, oral, rectal, intracloacal and/or intravaginal.
The term xe2x80x9csystemic immunityxe2x80x9d refers to antigen-specific host defense mediated by serum antibodies of IgM or IgG class or by activated T lymphocytes.
The term xe2x80x9cmucosal immunityxe2x80x9d refers to antigen-specific host defense mediated by antibodies of IgA class present in the host or secreted into the lumen of different organs.
The term xe2x80x9cmucosal vaccinexe2x80x9d refers to vaccines which are administered via a nonparenteral route to increase mucosal or systemic immune response to an antigen.
The term xe2x80x9cmucosal adjuvantxe2x80x9d refers to adjuvants which are administered via a nonparenteral route to increase mucosal or systemic immune response to an antigen.
The term xe2x80x9cgrafted polymerxe2x80x9d refers to polymers which are obtained by the addition of chemical groups with a significant effect on chemical, physicochemical or biological properties of the polymer.
The term xe2x80x9ccopolymerxe2x80x9d refers to polymers which are obtained by the polymerisation of two or more distinct monomers in conjunction with one another with significant distinct chemical, physiochemical or biological properties as compared to polymers obtained from either monomer.
The term xe2x80x9cliquid mediumxe2x80x9d refers to mediums of liquid including, but not limited to: aqueous solutions, physiological aqueous solutions, emulsions of the type oil-in-water and suspensions of insoluble salts in an aqueous solution (as well as other types of pharmaceutically-acceptable carriers).
The preferred liquid mediums are aqueous solutions with physiological aqueous solutions being most preferred, although one of the advantages of the present invention is that the vaccine formulation incorporating the mucosal adjuvants disclosed herein need not be physiologic.